High-stability EGCG (epigallocatechin gallate) microsphere emulsion for skin whitening as well as preparation method and application of high-stability EGCG microsphere emulsion
By preparing a microsphere emulsion formed by the self-assembly of co-amorphous material with collagen and BOXER, the problem of poor stability of EGCG was solved, achieving high stability and whitening effect in cosmetics.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGZHOU ZHONGZHUANG BEAUTY COSMETICS CO LTD
- Filing Date
- 2026-03-05
- Publication Date
- 2026-05-19
AI Technical Summary
The application of EGCG in cosmetics is hampered by its poor stability. It is easily affected by factors such as temperature, light, and pH, which can lead to molecular degradation, oxidation, and isomerization, thus affecting bioactivity and product quality.
By preparing a co-amorphous compound formed from EGCG, ergothionein, and 4-butylresorcinol, the solubility of EGCG in water is enhanced, and it self-assembles with collagen and bosine to form a microsphere emulsion, thus constructing a multi-molecular barrier to protect EGCG.
It improves the stability of EGCG, effectively inhibits tyrosinase activity, and has excellent whitening effects, making it suitable for the cosmetics industry.
Smart Images
Figure CN122056784A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomaterials technology, and in particular to a highly stable EGCG microsphere emulsion for skin whitening, its preparation method, and its application. Background Technology
[0002] Epigallocatechin gallate (EGCG) is the most abundant active monomer in green tea polyphenols. Due to its powerful antioxidant, anti-inflammatory, and specific tyrosinase-inhibiting abilities, it is widely recognized as a promising natural whitening agent and photoprotectant. However, EGCG's poor stability limits its direct application and efficacy in cosmetic products. The presence of multiple active phenolic hydroxyl groups in its molecular structure makes it highly sensitive to temperature, light, and pH, readily undergoing degradation, oxidation, and isomerization. This not only reduces or eliminates its biological activity but also causes product discoloration and spoilage, shortening shelf life.
[0003] Currently, various delivery strategies have been explored in existing technologies to improve the stability of EGCG. For example, lipid-based encapsulation strategies can improve the encapsulation efficiency of EGCG to some extent and reduce its direct contact with the external environment. However, the phospholipid bilayer structure of lipid carriers is prone to oxidation during long-term storage, making it difficult to effectively maintain the long-term activity of EGCG. While polymer nanoparticle-based encapsulation strategies can provide a better protective barrier for EGCG, these carriers generally have limited loading capacity and complex preparation processes, often requiring the use of organic solvents. This not only increases production costs and difficulty but may also introduce the risk of organic solvent residue, affecting product safety.
[0004] Therefore, developing a new strategy that is simple in process and can effectively protect EGCG is an urgent problem to be solved. Summary of the Invention
[0005] This invention provides a highly stable EGCG microsphere emulsion for skin whitening. The EGCG microsphere emulsion has a simple preparation process, can effectively protect EGCG, has high stability, and can effectively inhibit tyrosinase activity, thus exhibiting excellent whitening effects.
[0006] This invention also provides a method for preparing the above-mentioned highly stable EGCG microsphere emulsion for skin whitening. By preparing epigallocatechin gallate (EGCG), ergothioneine and 4-butylresorcinol into a co-amorphous compound, the solubility of EGCG in water is enhanced while the stability of EGCG is improved. The resulting EGCG microsphere emulsion has high stability and excellent whitening effect, and has broad application prospects in the cosmetics field.
[0007] This invention also provides the application of the above-mentioned highly stable EGCG microsphere emulsion for skin whitening in the preparation of cosmetic products. Research by this invention shows that the highly stable EGCG microsphere emulsion for skin whitening can effectively inhibit tyrosinase activity and has excellent whitening effects; therefore, it can be used in the preparation of cosmetic products.
[0008] The first aspect of the present invention provides a highly stable EGCG microsphere emulsion for skin whitening, wherein the raw materials for preparing the EGCG microsphere emulsion include: co-amorphous material, collagen, and β-Xylane;
[0009] The co-amorphous compound is made from epigallocatechin gallate, ergothioneine and 4-butylresorcinol.
[0010] In the highly stable EGCG microsphere emulsion for skin whitening described above, the mass ratio of epigallocatechin gallate, ergothioneine, and 4-butylresorcinol is 30:2:1.
[0011] The highly stable EGCG microsphere emulsion for skin whitening described above contains microspheres with an average particle size of 100-300 nm.
[0012] A second aspect of the present invention provides a method for preparing the highly stable EGCG microsphere emulsion for skin whitening, comprising the following steps:
[0013] S1. Epigallocatechin gallate, ergothioneine, 4-butylresorcinol and anhydrous ethanol are mixed and subjected to a first ultrasonic treatment. Excess solvent is removed by rotary evaporation under vacuum to obtain epigallocatechin gallate-ergothioneine-4-butylresorcinol co-amorphous product.
[0014] S2. The co-amorphous material is re-dissolved in water and subjected to a second ultrasonic treatment to obtain an aqueous solution of the co-amorphous material;
[0015] S3. Add the collagen aqueous solution to the amorphous material aqueous solution and stir for the first time, then add the BOXER aqueous solution and stir for the second time to obtain the EGCG microsphere emulsion.
[0016] In the preparation method of the highly stable EGCG microsphere emulsion for skin whitening as described above, in step S1, the first ultrasonic treatment has a power of 300-600W, a frequency of 30-60KHZ, and a time of 10-50min.
[0017] In the preparation method of the highly stable EGCG microsphere emulsion for skin whitening as described above, in step S1, the rotary evaporation temperature is 45-50℃ and the pressure is 400-600 mmHg.
[0018] In the preparation method of the highly stable EGCG microsphere emulsion for skin whitening as described above, in step S2, the mass-to-volume ratio of the co-amorphous material to the water is (40-80 mg): (0.5-5 mL).
[0019] And / or, in step S2, the second ultrasonic treatment has a power of 300-600W, a frequency of 30-60KHZ, and a time of 15-30min.
[0020] In the preparation method of the highly stable EGCG microsphere emulsion for skin whitening as described above, in step S3, the concentration of the collagen aqueous solution is 0.01-0.06 mg / mL, and the concentration of the BOXER aqueous solution is 20-60 mg / mL.
[0021] In the preparation method of the highly stable EGCG microsphere emulsion for skin whitening as described above, in step S3, the first stirring is: stirring at room temperature for 10-20 min;
[0022] And / or, in step S3, the second stirring is: stirring at room temperature for 10-20 minutes.
[0023] A third aspect of the present invention provides the application of an EGCG microsphere emulsion in the preparation of cosmetic products, wherein the EGCG microsphere emulsion is the highly stable EGCG microsphere emulsion for skin whitening described above or the highly stable EGCG microsphere emulsion for skin whitening prepared by the aforementioned preparation method.
[0024] The solution of the present invention has at least the following effects:
[0025] This invention provides a highly stable EGCG microsphere emulsion for skin whitening, the raw materials of which include: amorphous components, collagen, and BPOXY; wherein the amorphous components are made from epigallocatechin gallate, ergothioneine, and 4-butylresorcinol. This invention first prepares the amorphous components from epigallocatechin gallate, ergothioneine, and 4-butylresorcinol, enhancing the solubility of EGCG in water while improving its stability; then, the amorphous components are self-assembled with collagen and BPOXY to finally form an emulsion containing microspheres, namely the EGCG microsphere emulsion. This EGCG microsphere emulsion has a simple preparation process, effectively protects EGCG, exhibits high stability, and effectively inhibits tyrosinase activity, demonstrating excellent whitening effects and broad application prospects in the cosmetics field. Attached Figure Description
[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0027] Figure 1 This is a photograph of the EGCG emulsion used in Comparative Example 4 of the present invention.
[0028] Figure 2 The powder X-ray diffraction (XRD) patterns of the epigallocatechin gallate-ergothionein-4-butylresorcinol co-amorphous compound (co-amorphous compound) in Example 1 of the present invention and the epigallocatechin gallate-ergothionein-4-butylresorcinol mixture (physical mixture) in Comparative Example 4 are shown.
[0029] Figure 3 This is a photograph of the EGCG microsphere emulsion in Example 1 of the present invention after being accelerated at 50°C for 15 days.
[0030] Figure 4 This is a photograph of the EGCG emulsion in Comparative Example 1 of the present invention after being accelerated at 50°C for 15 days.
[0031] Figure 5 This is a photograph of the EGCG emulsion in Comparative Example 2 of the present invention after being accelerated at 50°C for 15 days.
[0032] Figure 6 This is a photograph of the EGCG emulsion in Comparative Example 3 of the present invention after being accelerated at 50°C for 15 days.
[0033] Figure 7 This is a TEM image of the EGCG microsphere emulsion in Example 1 of the present invention. Detailed Implementation
[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.
[0035] In the following description, the term "and / or" is used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural.
[0036] In this invention, the use of terms such as "first" and "second" is for distinguishing similar objects and not for describing a specific order or sequence, and therefore should not be construed as a limitation of this invention.
[0037] Those skilled in the art should understand that, in the following description of the embodiments of the present invention, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.
[0038] Those skilled in the art will understand that the numerical ranges in the embodiments of the present invention should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0039] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0040] The first aspect of the present invention provides a highly stable EGCG microsphere emulsion for skin whitening, wherein the raw materials for preparing the EGCG microsphere emulsion include: amorphous material, collagen, and β-xylene; wherein the amorphous material is made of epigallocatechin gallate, ergothionein, and 4-butylresorcinol.
[0041] The present invention does not impose any particular limitation on the source of the above-mentioned raw materials, which can be purchased from commercial channels or prepared using methods known in the art.
[0042] In this invention, ergothionein and 4-butylresorcinol are used to form a co-amorphous compound with epigallocatechin gallate (EGCG), which enhances the solubility of EGCG in water and improves the stability of EGCG.
[0043] This invention involves the self-assembly of amorphous materials with collagen and β-hydroxyl group to form an emulsion containing microspheres, namely EGCG microsphere emulsion. The preparation process of this EGCG microsphere emulsion is simple, it can effectively protect EGCG, has high stability, and can effectively inhibit tyrosinase activity, thus exhibiting excellent whitening effects.
[0044] In one specific embodiment, the mass ratio of the epigallocatechin gallate, the ergothionein, and the 4-butylresorcinol is 30:2:1.
[0045] When the mass ratio of the epigallocatechin gallate (EGCG), the ergothioneine, and the 4-butylresorcinol meets the above conditions, the epigallocatechin gallate-ergothioneine-4-butylresorcinol co-amorphous compound (hereinafter referred to as co-amorphous compound) can be successfully prepared. The principle and function of preparing epigallocatechin gallate-ergothioneine-4-butylresorcinol co-amorphous compound according to the present invention are explained as follows: The co-amorphization of EGCG, ergothioneine and 4-butylresorcinol can destroy the crystal structure, form intermolecular hydrogen bonds and π-π stacking, and the resulting co-amorphous compound is in a high-energy disordered state, which changes the crystal stacking mode of EGCG, reduces the lattice energy, and thus improves the apparent solubility of EGCG. At the same time, the polyphenolic hydroxyl groups of EGCG, the sulfur-nitrogen heterocycle of ergothioneine and the phenolic hydroxyl groups of 4-butylresorcinol form complementary intermolecular forces, which effectively reduce molecular migration rate, delay phase separation and improve the stability of EGCG microsphere emulsion.
[0046] In one specific embodiment, the EGCG microsphere emulsion contains microspheres, and the average particle size of the microspheres is 100-300 nm.
[0047] The principle and function of preparing microspheres in this invention are explained as follows: The co-amorphous material is re-dissolved in water to dissolve EGCG, and then collagen and BOXIN are added sequentially. Microspheres are formed layer by layer through electrostatic interaction, which constructs multiple molecular barriers that can more effectively isolate external factors such as light, oxygen, and moisture, effectively protect EGCG, and thus further improve the stability of EGCG.
[0048] A second aspect of the present invention provides a method for preparing the highly stable EGCG microsphere emulsion for skin whitening, comprising the following steps:
[0049] S1. Epigallocatechin gallate, ergothioneine, 4-butylresorcinol and anhydrous ethanol are mixed and subjected to a first ultrasonic treatment. Excess solvent is removed by rotary evaporation under vacuum to obtain epigallocatechin gallate-ergothioneine-4-butylresorcinol co-amorphous product.
[0050] S2. The co-amorphous material is re-dissolved in water and subjected to a second ultrasonic treatment to obtain an aqueous solution of the co-amorphous material;
[0051] S3. Add the collagen aqueous solution to the amorphous material aqueous solution and stir for the first time, then add the BOXER aqueous solution and stir for the second time to obtain the EGCG microsphere emulsion.
[0052] Specifically, the present invention first mixes epigallocatechin gallate (EGCG), ergothioneine, 4-butylresorcinol, and a solvent, and then performs a first ultrasonic treatment to uniformly disperse the raw materials, obtaining an epigallocatechin gallate-ergothioneine-4-butylresorcinol suspension; then, the suspension is rotary evaporated to remove excess solvent, obtaining an epigallocatechin gallate-ergothioneine-4-butylresorcinol co-amorphous compound; next, the co-amorphous compound is reconstituted in water and subjected to a second ultrasonic treatment to fully dissolve the co-amorphous compound in water, obtaining an aqueous solution of the co-amorphous compound; then, the aqueous solution of the co-amorphous compound is added to a collagen aqueous solution and stirred for a first time, and then an aqueous solution of Bosein is added and stirred for a second time, so that EGCG is sequentially assembled with collagen and Bosein layer by layer, finally obtaining an EGCG microsphere emulsion.
[0053] The EGCG microsphere emulsion prepared by the method provided in this invention can effectively protect EGCG, has high stability, and can effectively inhibit tyrosinase activity, thus exhibiting excellent whitening effects.
[0054] In one specific embodiment, in step S1, the power of the first ultrasonic treatment is 300-600W, the frequency is 30-60KHZ, and the time is 10-50min.
[0055] When the power parameters in the first ultrasonic treatment meet the above conditions, mechanical vibration can be enhanced, solvent penetration can be accelerated, thereby improving dissolution efficiency and avoiding local overheating and component loss of raw materials (epigallocatechin gallate, ergothioneine, 4-butylresorcinol) due to excessive power. At the same time, the mass transfer process can be optimized and the dissolution effect can be improved at a frequency of 30-60KHZ.
[0056] In one specific embodiment, in step S1, the temperature of the rotary evaporation is 45-50°C and the pressure is 400-600 mmHg.
[0057] In this invention, the rotary evaporation temperature (45-50℃) combined with a vacuum environment (400-600mmHg) can accelerate the evaporation of excess solvent (anhydrous ethanol), while avoiding the decomposition or denaturation of heat-sensitive components caused by high temperature, and suppressing the risk of boiling over, reducing product sputtering loss caused by rapid vaporization of anhydrous ethanol.
[0058] In one specific embodiment, in step S2, the mass-to-volume ratio of the co-amorphous material to the water is (40-80 mg): (0.5-5 mL).
[0059] When the mass-to-volume ratio of the amorphous material to the water is within the above range, the amorphous material can be quickly dissolved and dispersed. Because the amorphous material particles are small and have a large specific surface area, they are more easily penetrated and dispersed by water during resolution, thus avoiding excessive dilution that leads to a low concentration of the effective ingredients.
[0060] In one specific embodiment, in step S2, the second ultrasonic treatment has a power of 300-600W, a frequency of 30-60KHZ, and a time of 15-30min.
[0061] When the power, frequency, and time parameters in the second ultrasonic treatment are each within the above range, the intermolecular hydrogen bonding can be enhanced, the stability of the amorphous region structure can be improved, and the disordered expansion of the amorphous region can be suppressed.
[0062] In one specific embodiment, in step S3, the concentration of the collagen aqueous solution is 0.01-0.06 mg / mL, and the concentration of the bosine aqueous solution is 20-60 mg / mL.
[0063] When the concentrations of the collagen aqueous solution and the BOXER aqueous solution are each within the above-mentioned range, it helps to form a uniform and stable emulsion, prevents stratification, and avoids droplet aggregation at high concentrations. BOXER, by forming a weak hydrogen bond network with the hydroxyl and carboxyl groups of collagen, can enhance the conformational stability and interfacial coverage density of collagen molecules, jointly promoting the formation of a stable EGCG microsphere emulsion.
[0064] In one specific embodiment, in step S3, the first stirring is: stirring at room temperature for 10-20 minutes.
[0065] In one specific embodiment, in step S3, the second stirring is: stirring at room temperature for 10-20 minutes.
[0066] A third aspect of this invention provides the application of an EGCG microsphere emulsion in the preparation of cosmetic products. The EGCG microsphere emulsion is either the highly stable EGCG microsphere emulsion for skin whitening described above, or a highly stable EGCG microsphere emulsion for skin whitening prepared by the aforementioned method. Research by this invention shows that this highly stable EGCG microsphere emulsion for skin whitening can effectively inhibit tyrosinase activity and has excellent whitening effects; therefore, it can be used in the preparation of cosmetic products.
[0067] The embodiments of the present invention will be described in detail below.
[0068] Example 1
[0069] The EGCG microsphere emulsion provided in this embodiment is obtained by a preparation method including the following process:
[0070] (1) 600 mg of epigallocatechin gallate (EGCG), 40 mg of ergothioneine, 20 mg of 4-butylresorcinol and 10 mL of anhydrous ethanol were added to a reaction vessel and sonicated for 30 min at a power of 500 W and a frequency of 40 kHz. After mixing evenly, an epigallocatechin gallate-ergothioneine-4-butylresorcinol suspension (hereinafter referred to as suspension) was obtained.
[0071] (2) The suspension was transferred to a rotary evaporator and rotary evaporated under vacuum (500 mmHg) and 50°C to completely remove excess solvent, to obtain epigallocatechin gallate-ergothionein-4-butylresorcinol co-amorphous compound (hereinafter referred to as co-amorphous compound).
[0072] (3) Dissolve 600 mg of the co-amorphous material in 10 mL of ultrapure water and sonicate it for 20 min at a power of 500 W and a frequency of 40 kHz to obtain an aqueous solution of the co-amorphous material.
[0073] (4) Collagen was dissolved in ultrapure water to prepare a collagen aqueous solution with a concentration of 0.04 mg / mL; and Brønsted was dissolved in ultrapure water to prepare a Brønsted was aqueous solution with a concentration of 40 mg / mL.
[0074] 20 mL of collagen aqueous solution was added to 10 mL of the co-amorphous material aqueous solution under stirring. The mixture was stirred at room temperature for 20 min, and then 10 mL of BOXER aqueous solution was added. The mixture was stirred at room temperature for another 20 min to obtain a transparent EGCG microsphere emulsion.
[0075] Comparative Example 1 (without ergothioneine)
[0076] The EGCG emulsion provided in this comparative example is essentially the same as that in Example 1, except that ergothioneine was not added. Specifically, the EGCG emulsion provided in this comparative example was obtained by a preparation method including the following steps:
[0077] (1) 600 mg of epigallocatechin gallate (EGCG), 20 mg of 4-butylresorcinol and 10 mL of anhydrous ethanol were added to a reaction vessel and sonicated for 30 min at a power of 500 W and a frequency of 40 kHz. After mixing evenly, an epigallocatechin gallate-4-butylresorcinol suspension was obtained.
[0078] (2) The suspension was transferred to a rotary evaporator and rotary evaporated under vacuum (500 mmHg) and 50°C to completely remove excess solvent and obtain the product;
[0079] (3) Dissolve 600 mg of the product in 10 mL of ultrapure water and sonicate it for 20 min at a power of 500 W and a frequency of 40 kHz to obtain an aqueous solution of the product.
[0080] (4) Collagen was dissolved in ultrapure water to prepare a collagen aqueous solution with a concentration of 0.04 mg / mL; and Brønsted was dissolved in ultrapure water to prepare a Brønsted was aqueous solution with a concentration of 40 mg / mL.
[0081] 20 mL of collagen aqueous solution was added to 10 mL of the product aqueous solution under stirring. The mixture was stirred at room temperature for 20 min, and then 10 mL of BOXER aqueous solution was added. The mixture was stirred at room temperature for another 20 min to obtain a transparent EGCG emulsion.
[0082] Comparative Example 2 (without added collagen)
[0083] The EGCG emulsion provided in this comparative example is essentially the same as that in Example 1, except that collagen was not added. Specifically, the EGCG emulsion provided in this comparative example was obtained by a preparation method including the following steps:
[0084] (1) 600 mg of epigallocatechin gallate (EGCG), 40 mg of ergothioneine, 20 mg of 4-butylresorcinol and 10 mL of anhydrous ethanol were added to a reaction vessel and sonicated for 30 min at a power of 500 W and a frequency of 40 kHz. After mixing evenly, an epigallocatechin gallate-ergothioneine-4-butylresorcinol suspension was obtained.
[0085] (2) The suspension was transferred to a rotary evaporator and rotary evaporated under vacuum (500 mmHg) and 50°C to completely remove excess solvent, to obtain epigallocatechin gallate-ergothionein-4-butylresorcinol co-amorphous compound (hereinafter referred to as co-amorphous compound).
[0086] (3) Dissolve 600 mg of the co-amorphous material in 10 mL of ultrapure water and sonicate it for 20 min at a power of 500 W and a frequency of 40 kHz to obtain an aqueous solution of the co-amorphous material.
[0087] (4) Dissolve bosine in ultrapure water to prepare a bosine aqueous solution with a concentration of 40 mg / mL;
[0088] 10 mL of a Bosein aqueous solution was added to 10 mL of the co-amorphous material aqueous solution under stirring, and stirred at room temperature for 20 min to obtain a transparent EGCG emulsion.
[0089] Comparative Example 3 (without added Bosonicin)
[0090] The EGCG emulsion provided in this comparative example is essentially the same as that in Example 1, except that collagen was not added. Specifically, the EGCG emulsion provided in this comparative example was obtained by a preparation method including the following steps:
[0091] (1) 600 mg of epigallocatechin gallate (EGCG), 40 mg of ergothioneine, 20 mg of 4-butylresorcinol and 10 mL of anhydrous ethanol were added to a reaction vessel and sonicated for 30 min at a power of 500 W and a frequency of 40 kHz. After mixing evenly, an epigallocatechin gallate-ergothioneine-4-butylresorcinol suspension was obtained.
[0092] (2) The suspension was transferred to a rotary evaporator and rotary evaporated under vacuum (500 mmHg) and 50°C to completely remove excess solvent, to obtain epigallocatechin gallate-ergothionein-4-butylresorcinol co-amorphous compound (hereinafter referred to as co-amorphous compound).
[0093] (3) Dissolve 600 mg of the co-amorphous material in 10 mL of ultrapure water and sonicate it for 20 min at a power of 500 W and a frequency of 40 kHz to obtain an aqueous solution of the co-amorphous material.
[0094] (4) Collagen was dissolved in ultrapure water to prepare a collagen aqueous solution with a concentration of 0.04 mg / mL;
[0095] 20 mL of collagen aqueous solution was added to 10 mL of the aforementioned amorphous material aqueous solution under stirring, and stirred at room temperature for 20 min to obtain a transparent EGCG emulsion.
[0096] Comparative Example 4 (no co-amorphous material formed)
[0097] The EGCG emulsion provided in this comparative example was obtained by a preparation method including the following process:
[0098] (1) Mix 600 mg of epigallocatechin gallate (EGCG), 40 mg of ergothioneine, and 20 mg of 4-butylresorcinol evenly to obtain an epigallocatechin gallate-ergothioneine-4-butylresorcinol mixture (hereinafter referred to as physical mixture).
[0099] (2) Add 600 mg of the above-mentioned epigallocatechin gallate-ergothionein-4-butylresorcinol mixture to 10 mL of ultrapure water, and sonicate for 20 min under the conditions of 500 W power and 40 KHZ frequency. After mixing evenly, a mixture solution is obtained.
[0100] (3) Collagen was dissolved in ultrapure water to prepare a collagen aqueous solution with a concentration of 0.04 mg / mL; and Brønsted was dissolved in ultrapure water to prepare a Brønsted solution with a concentration of 40 mg / mL.
[0101] Add 20 mL of collagen aqueous solution to 10 mL of the mixture while stirring, stir at room temperature for 20 min, then add 10 mL of BOXER aqueous solution, and continue stirring at room temperature for 20 min to obtain EGCG emulsion.
[0102] Figure 1 This is a photograph of the EGCG emulsion used in Comparative Example 4 of this invention. Figure 1 It is known that solid particles precipitate out of the EGCG emulsion, indicating that it is inherently unstable, and therefore no further stability testing will be conducted.
[0103] Performance testing
[0104] 1. Powder X-ray diffraction (XRD) test
[0105] Powder X-ray diffraction (XRD) was performed on the epigallocatechin gallate-ergothioneine-4-butylresorcinol co-amorphous compound (co-amorphous compound) in Example 1 of the present invention and the epigallocatechin gallate-ergothioneine-4-butylresorcinol mixture (physical mixture) in Comparative Example 1. The test conditions were as follows: Cu target (40 kV, 40 mV), 2θ angle, step scan 0.026° / step, scan range 5.00°~90.00°, scan rate 10° / min. The results are as follows. Figure 2 As shown.
[0106] Figure 2 The results showed that the diffraction peaks of the physical mixture were sharp, with several strong crystalline diffraction peaks between 10° and 36°; while in the spectrum of the co-amorphous compound, the co-amorphous compound exhibited diffuse diffraction peaks, with several obvious diffraction peaks disappearing at 10.5°, 12°, 17°, 19.1°, 20° and 25.8°, indicating that epigallocatechin gallate (EGCG), ergothioneine and 4-butylresorcinol coexisted in an amorphous state, and the epigallocatechin gallate-ergothioneine-4-butylresorcinol co-amorphous compound was successfully prepared.
[0107] 2. Stability Test
[0108] The EGCG microsphere emulsion in Example 1 and the EGCG emulsions in Comparative Examples 1-3 were used as test samples for stability testing. The specific test method is as follows: the test samples were stored in a constant temperature chamber at 50°C for 15 days, the state of the test samples was observed and photographed, and the recorded results were summarized in Table 1. Figure 3This is a photograph of the EGCG microsphere emulsion in Example 1 of the present invention after being accelerated at 50°C for 15 days. Figure 4 This is a photograph of the EGCG emulsion in Comparative Example 1 of the present invention after being accelerated at 50°C for 15 days. Figure 5 This is a photograph of the EGCG emulsion in Comparative Example 2 of the present invention after being accelerated at 50°C for 15 days. Figure 6 This is a photograph of the EGCG emulsion in Comparative Example 3 of the present invention after being accelerated at 50°C for 15 days.
[0109] Table 1 Stability Test Results
[0110]
[0111] Depend on Figures 3-6 As shown in Table 1, the EGCG microsphere emulsion of Example 1 showed no abnormalities after 15 days of accelerated treatment at 50°C, demonstrating high stability; while the EGCG emulsions of Comparative Examples 1-3 showed varying degrees of yellowing after 15 days of accelerated treatment at 50°C, indicating instability.
[0112] 3. Transmission electron microscopy (TEM) testing
[0113] TEM testing was performed on the EGCG microsphere emulsion in Example 1 of this invention, and the results are as follows: Figure 7 As shown. Figure 7 This is a TEM image of the EGCG microsphere emulsion in Example 1 of the present invention.
[0114] Depend on Figure 7 It is known that the EGCG microsphere emulsion in Example 1 has a microsphere structure with an average particle size of about 150 nm.
[0115] 4. Whitening efficacy test
[0116] The whitening efficacy of the EGCG microsphere emulsion in Example 1 of this invention was tested, with the epigallocatechin gallate aqueous solution (EGCG aqueous solution) as the reference standard. The specific steps are as follows:
[0117] Frozen mouse melanoma cells (B16 cells, Wuhan Pronosai Biotechnology Co., Ltd.) were retrieved from the liquid nitrogen tank and thawed. When the cells reached 70%-80% confluence, they were digested and counted. Cells were then distributed at 8 × 10⁸ cells per well. 4Cells were seeded at a density of 1000 mg / L into 12-well plates, and then the seeded 12-well plates were incubated at 37°C and 5% CO2 for 24 h. Control, model and sample groups were set up, with 1 mL of drug administered to each well: the control group was given DMEM medium containing 2% FBS, the model group was given DMEM medium containing 200 nmol / L α-MSH and 2% FBS, and the sample group was given DMEM medium containing 200 nmol / L α-MSH, different concentrations of sample and 2% FBS. Each group was set up with 3 replicates. After drug administration, the wells were incubated at 37°C and 5% CO2 for 48 h. See Table 2 for details.
[0118] Experimental reagents: DMEM medium (Gibco); fetal bovine serum (FBS, Zhejiang Tianhang Biotechnology Co., Ltd.); PBS (Solepro), trypsin (Gibco), α-melanocyte-stimulating hormone (α-MSH, APEX); PBS (Solepro); Triton X-100 (Solepro); L-DOPA (Solepro).
[0119] Table 2 Experimental Scheme
[0120]
[0121] After culturing for 48 h, the supernatant was discarded, and the cells were washed with PBS. 400 μL of cell lysis buffer containing 1% Triton X-100 was added to each well, and the cells were lysed at -80℃ for 30 min. The lysed cell lysate was collected, centrifuged (5000 rpm, 5 min, 4℃), and 100 μL of the supernatant was transferred to a 96-well plate. 100 μL of 0.1% (w / v) L-DOPA solution was added, and the plates were incubated at 37℃ for 1 h. The absorbance (OD value) was measured at 495 nm using a microplate reader. Statistical analysis was performed using GraphPad Prism 9.0 software, and the significance was analyzed using the t-test. The experimental results are shown in Table 3. Tyrosinase activity = (OD value of sample group / OD value of control group) × 100%; Tyrosinase inhibition rate = (|tyrosinase activity of sample group - tyrosinase activity of model group|) / tyrosinase activity of model group × 100%.
[0122] Table 3 Experimental Results
[0123]
[0124] Table 3 shows that, compared with the control group, the tyrosinase activity value of the model group was significantly increased, indicating that the α-MSH-induced mouse melanoma cell validation model was successfully established. Compared with the model group, the tyrosinase activity value of the control product EGCG decreased by 3.32%; the tyrosinase activity value of the EGCG microsphere emulsion in Example 1 decreased significantly by 16.44%. Compared with the control product EGCG, the EGCG microsphere emulsion provided in this embodiment of the invention can effectively inhibit tyrosinase activity, has excellent whitening effect, and has broad application prospects in the cosmetics field.
[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A highly stable EGCG microsphere emulsion for skin whitening, characterized in that, The raw materials for preparing the EGCG microsphere emulsion include: co-amorphous material, collagen, and β-Xylane; The co-amorphous compound is made from epigallocatechin gallate, ergothioneine and 4-butylresorcinol.
2. The highly stable EGCG microsphere emulsion for skin whitening according to claim 1, characterized in that, The mass ratio of the epigallocatechin gallate, the ergothioneine, and the 4-butylresorcinol is 30:2:
1.
3. The highly stable EGCG microsphere emulsion for skin whitening according to claim 1, characterized in that, The EGCG microsphere emulsion contains microspheres with an average particle size of 100-300 nm.
4. A method for preparing a highly stable EGCG microsphere emulsion for skin whitening according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Epigallocatechin gallate, ergothioneine, 4-butylresorcinol and anhydrous ethanol are mixed and subjected to a first ultrasonic treatment. Excess solvent is removed by rotary evaporation under vacuum to obtain epigallocatechin gallate-ergothioneine-4-butylresorcinol co-amorphous product. S2. The co-amorphous material is re-dissolved in water and subjected to a second ultrasonic treatment to obtain an aqueous solution of the co-amorphous material; S3. Add the collagen aqueous solution to the amorphous material aqueous solution and stir for the first time, then add the BOXER aqueous solution and stir for the second time to obtain the EGCG microsphere emulsion.
5. The method for preparing the highly stable EGCG microsphere emulsion for skin whitening according to claim 4, characterized in that, In step S1, the power in the first ultrasonic treatment is 300-600W, the frequency is 30-60KHZ, and the time is 10-50min.
6. The method for preparing the highly stable EGCG microsphere emulsion for skin whitening according to claim 4, characterized in that, In step S1, the temperature of the rotary evaporation is 45-50℃ and the pressure is 400-600 mmHg.
7. The method for preparing the highly stable EGCG microsphere emulsion for skin whitening according to claim 4, characterized in that, In step S2, the mass-to-volume ratio of the amorphous material to the water is (40-80 mg): (0.5-5 mL). And / or, in step S2, the second ultrasonic treatment has a power of 300-600W, a frequency of 30-60KHZ, and a time of 15-30min.
8. The method for preparing the highly stable EGCG microsphere emulsion for skin whitening according to claim 4, characterized in that, In step S3, the concentration of the collagen aqueous solution is 0.01-0.06 mg / mL, and the concentration of the bosine aqueous solution is 20-60 mg / mL.
9. The method for preparing the highly stable EGCG microsphere emulsion for skin whitening according to claim 4, characterized in that, In step S3, the first stirring is: stirring at room temperature for 10-20 minutes; And / or, in step S3, the second stirring is: stirring at room temperature for 10-20 minutes.
10. The application of an EGCG microsphere emulsion in the preparation of cosmetic products, characterized in that, The EGCG microsphere emulsion is the highly stable EGCG microsphere emulsion for skin whitening as described in any one of claims 1-3, or the highly stable EGCG microsphere emulsion for skin whitening prepared by the preparation method described in any one of claims 4-9.